In vitro callus induction of Plukenetia Volubilis (Sacha Inchi), a PUFA-rich plant

The young leaves and ovules of Plukenetia volubilis were cultured in MS medium under 5 different treatments of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) incubated in different conditions, either 24 hours light or 24 hours dark condition to develop an in vitro micropropagation through callus induction. Despite a series of contamination after surface sterilization, the young leaves explants deemed promising in developing callus in media supplemented with both plant growth regulators of 0.1 mg L-1 2,4-D + 0.05 mg L-1 BAP and 1.0 mg L-1 2,4-D + 0.05 mg L-1 BAP. Interestingly, it is the similar concentration that yielded callus from ovule. All treatments developed callus from ovule, in which the best callus was induced from MS media supplemented with 0.1 mg L-1 2,4- D + 0.05 mg L-1 BAP in both light and dark conditions compared to the single use of 2,4-D. This is the first report attempted on using ovules of sacha inchi as explant

In silico identification and characterization of Kaurene Synthase protein in Stevia rebaudiana MS007

Stevia rebaudiana (Sr), belonging to Asteraceae family is a plant native to Paraguay. It is currently being used as a healthier alternative for sugar. Sr produces steviol glycosides (SGs), a group of secondary metabolite compounds that is responsible for its sweetening taste. SGs act as sweetener due to the presence of two major compounds, Stevioside and Rebaudioside A. Biosynthesis of these compounds involve enzymes such as geranylgeranyl pyrophosphate (GPPS), copalyl diphosphate synthase (CPPS), kaurene synthase (KS) and kaurene oxidase (KO) in the pathway. In this study, the identification and characterization of Stevia rebaudiana MS007 kaurene synthase (SrKS) were done by in silico analysis of the transcriptomic dataset. Homology search from BLASTx resulting in SrKSfrom query Cluster-31069.42907 (Sr MS007) of transcriptomic dataset shows the highest similarity percentage identity (99.62%). ExPasy tools were used to translate the nucleotide sequence into protein sequence. The protein domain is predicted by protein domain search analysis using Interpro and shows IPR005630 (terpene synthase metal-binding domain) available at positions 454 to 719 and IPR001906 (terpene-synthase-N-terminal-domain) at position 222 to 411 as the domains. In constructing the phylogenetic analysis tree, multiple sequence alignment was initially done using MUSCLE and MEGA-X was used as phylogenetic tree analysis tools. Cluster-31069.42907 shows the relationship between the ancestors, based on the bootstrap value. Bootstrap value of Helianthus annuus and Stevia rebaudiana is 100% as both the sequences are from the Asteraceae family. This study contributes to a deeper understanding of S. rebaudiana MS007 Kaurene synthase through in silico analysis

Optimisation of culture condition for Sacha Inchi (Plukenetia volubilis) Callus induction

Plukenetia volubilis or commonly known as sacha inchi produces wide range of health‐promoting bioactive compounds. The plant has large edible seeds that are rich in phenolics, minerals and essential fatty acid, such as omega 3, omega 6, omega 7 and omega 9. In vitro cultures could serve as alternative in producing many essential sacha inchi bioactive compounds. In this study, as an initial step towards initiating in vitro cultures, the effect of 2,4-D and TDZ on callus induction using leaves and male flower as explants were investigated. Surface sterilization of the sacha inchi explants were done by using 70% ethanol (30 seconds) and 0.5% sodium chloride (8 minutes) to overcome culture contamination. The sterilization method resulted in 82.5% and 95% survival rate for leaf and flower explants, respectively. Next, for callus induction the explants were cultured on MS medium supplemented with different concentrations of 2,4-D and TDZ, either alone or in combination and grown in 24 hours dark photoperiods. The morphology and size of callus obtained varied according to the treatment. Callus produced are either friable or compact with either creamy white, pure white or brownish colour. For both explants, the best response in term of callus size, friability and creamy white callus was obtained when cultured on MS medium supplemented with 3% (w/v) sucrose in combination with 1.0 mg/L 2,4-D and 0.005 mg/L TDZ

Callus induction and identification of DNA variation in callus derived from Etlingera elatior in vitro culture using ISSR marker

Etlingera elatior or torch ginger is a promising horticultural plant with various economic values that has been used for medicinal, culinary, and ornamental purposes in many countries. The extravagant and showy inflorescence has made E. elatior a valuable plant that can be used for cut flower and floral decoration. However, the plant itself lack of genetic variety with narrow genetic base due to its nature as an asexual propagated plant. To overcome this problem, somaclonal variation arises from the in vitro culture can be used to overcome the lack of variation in E. elatior. Moreover, early detection of the variation in callus stage using ISSR markers will help to examine the genetic variability of the induced callus. For this project, an optimum sterilization technique with contamination rate of 5% has been successfully developed. Furthermore, white friable calli were successfully developed from the innermost part of young closed buds. The results showed that the Murashige and Skoog medium supplemented with 30 g/L glucose, 3 mg/L 2, 4-D and 1.5 mg/L BAP has the highest percentage of callus induction (50%) after 20 weeks of culture. The calli were transferred into shoot induction media with different concentrations of BAP, NAA and TDZ. The calli from the 11 different media were evaluated for their genetic variations by seven primers of ISSR markers. A total of 72 bands were generated of which 51 were polymorphic with mean percentage of polymorphic bands was 72%. From our results, the calli were affected by various concentrations of auxin and cytokinin for callus and shoot induction treatments. In short, ISSR marker successfully revealed the occurrence of genetic variations in the induced callus. Even though, no in vitro shoots were successfully regenerated, this study revealed that there is a potential to generate new variants through in vitro culture

De novo transcriptome dataset of Stevia rebaudiana accession MS007

Stevia rebaudiana (S. rebaudiana) is a herbaceous and perennial plant belonging to Asteraceae family. The genus stevia is well known as a natural producer of sweetener comprising non-caloric and non-carcinogenic steviol glycosides. In recent years, the capability in producing natural sweetner has increased the demand for S. rebaudiana as substitute of processed sugars. Flowering phase of S. rebaudiana has shown to affect the content of steviol glycosides in the leaves. Steviol glycosides level is the highest at the time of flower bud formation and lowest at time preceding and following flower bud formation. Therefore, sequencing and analysing the genes that are involved in flowering phase will provide platform for gene manipulation in increasing steviol glycosides content. The Stevia transcriptome data that include two stages of growth (before flowering and after flowering), were obtained using Illumina RNA-seq technology and can be accessed at NCBI Sequence Read Archive under Accession No. SRX6362785 and SRX6362784

Transcriptome profiling of stevia rebaudiana ms007 revealed genes involved in flowedevelopment

Stevia rebaudiana is a medicinal plant recommended to diabetic or obese patients as an alternative sweetener owing to its low-calorie property. Previous studies have found that the stevioside level is highest at the time of flower bud formation and lowest at the time of preceding and following flower bud formation. Hence, this study aims to identify the genes involved in the flowering of local S. rebaudiana accession MS007 by investigating the transcriptomic data of two stages of growth, before flowering (BF) and after flowering (AF) that were deposited under accession number SRX6362785 and SRX6362784 at the NCBI SRA database. The transcriptomic study managed to annotate 108299 unigenes of S. rebaudiana with 8871 and 9832 genes that were differentially expressed in BF and AF samples, respectively. These genes involved in various metabolic pathways related to flower development, response to stimulus as well as photosynthesis. Pheophorbide A oxygenase (PAO), eukaryotic translation initiation factor 3 subunit E (TIF3E1), and jasmonate ZIM domain-containing protein 1 (JAZ1) were found to be involved in the flower development. The outcome of this study will help further research in the manipulation of the flowering process, especially in the breeding programme to develop photo-insensitive Stevia plant